Fig. 6

Fig. 6 miRNAs Establish the Right-Handed Dominance of the BMP Pathway Leading to Heart Laterality in Vertebrates (A) Luciferase reporter assay for the mouse Snail1 3′UTR in the presence of wild-type miRNAs (WT seed) and miRNAs with mutated seed sequence (seed mut). Analysis of miRNAs with WT seed together with 3′ UTR with mutations on the MRE (MRE mut), was compared to that with the wild-type 3′ UTRs. Data represent mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, not significant, two-sided Student’s t test WT seed versus seed mut, or MRE mut, n = 4 independent experiments. (B) qPCR for mature miRNAs from E8.5 mouse embryos dissected in left and right halves. Data represent mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01, two-sided Student’s t test left versus right halves, n = 4 independent pools of 6 halves per pool. (C) E8.5 mouse embryos were embedded and cryosectioned. Mature miRNA ISH was performed in transverse sections revealing higher miR-34a and miR-92a expression on the left-hand side (arrowheads), including the LPM. NT, neural tube; LPM, lateral plate mesoderm; som, somite. Scale bar: 100 μm. miR-16 is included as a control of a microRNA expressed in the in a bilateral symmetric manner, including the LPM. (D) EGFP transcription driven by the regulatory elements of prrx1a in transgenic zebrafish show bilaterally symmetric expression at the time when both transcripts and protein levels are L/R asymmetric (blue arrowheads in sections). Quantification of prrx1a area and width of signal is shown. Data represent interquartile distribution with 10–90 percentile, ∗∗∗ p < 0.001, two-sided Student’s t test prrx1a versus EGFP, n = 25 embryos per condition. Scale bar: 250 μm. (E) BMP signaling measured as p-SMAD1/5/8 protein expression shows bilateral symmetry. Scale bar: 250 μm. (F) In situ hybridization for Snail1 in E8.5 wt embryos show asymmetric expression in the right LPM (7/13, insets magnified below; LLPM/RLPM, left/right lateral plate mesoderm). EGFP transcripts in E8.5 transgenic (Tg) embryos show symmetric expression in the LPM (11/11, insets below). Scale bar: 100 μm. See also Figure S5.